Device, systems and methods for analyzing a target analyte

ABSTRACT

This disclosure is directed to a cap for obtaining a target analyte from a suspension. The cap introduces a magnetic field or a magnetic gradient to the tube to draw the target analyte bound to a particle to the cap. In one aspect, a cap includes a magnetic insert and a receiving piece. The magnetic insert includes a stopper and a magnet extending from the stopper; and, the receiving piece, which is configured to hold the magnetic insert, includes a receiving stopper and a sheath. The sheath may include imaging slides on opposite sides of the sheath. In another aspect, the cap may include a stopper and an embedded magnet. The cap may include an analysis piece on a bottom end of the stopper. In yet another aspect, the cap may include a fluid compartment and a filter at a bottom end of the stopper.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of Provisional Application No.61/803,340, filed Mar. 19, 2013.

TECHNICAL FIELD

This disclosure relates generally to density-based fluid separation and,in particular, to systems and methods the separation, axial expansion ofconstituent suspension fractions layered by centrifugation, and analysisof a target analyte.

BACKGROUND

Suspensions often include materials of interest that are difficult todetect, extract and isolate for analysis. For instance, whole blood is asuspension of materials in a fluid. The materials include billions ofred and white blood cells and platelets in a proteinaceous fluid calledplasma. Whole blood is routinely examined for the presence of abnormalorganisms or cells, such as fetal cells, endothelial cells, epithelialcells, parasites, bacteria, and inflammatory cells, and viruses,including HIV, cytomegalovirus, hepatitis C virus, and Epstein-Barrvirus and nucleic acids. Currently, practitioners, researchers, andthose working with blood samples try to separate, isolate, and extractcertain components of a peripheral blood sample for examination. Typicaltechniques used to analyze a blood sample include the steps of smearinga film of blood on a slide and staining the film in a way that enablescertain components to be examined by bright field microscopy.

On the other hand, materials of interest composed of particles thatoccur in very low numbers are especially difficult if not impossible todetect and analyze using many existing techniques. Consider, forinstance, circulating tumor cells (“CTCs”), which are cancer cells thathave detached from a tumor, circulate in the bloodstream, and may beregarded as seeds for subsequent growth of additional tumors (i.e.,metastasis) in different tissues. The ability to accurately detect andanalyze CTCs is of particular interest to oncologists and cancerresearchers, but CTCs occur in very low numbers in peripheral wholeblood samples. For instance, a 7.5 ml sample of peripheral whole bloodthat contains as few as 3 CTCs is considered clinically relevant in thediagnosis and treatment of a cancer patient. However, detecting even 1CTC in a 7.5 ml blood sample may be clinically relevant and isequivalent to detecting 1 CTC in a background of about 40-50 billion redand white blood cells. Using existing techniques to find, isolate andextract as few as 3 CTCs of a whole blood sample is extremely timeconsuming, costly and is extremely difficult to accomplish.

As a result, practitioners, researchers, and those working withsuspensions continue to seek systems and methods to more efficiently andaccurately detect, isolate and extract target materials of a suspension.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show exploded views of example magnetic caps.

FIG. 2 shows an exploded view of an example magnetic cap.

FIGS. 3A-3E show example magnetic caps.

FIGS. 4A-4C show example magnetic caps.

FIGS. 5A-5B show isometric views of two example tube and float systems.

FIG. 5C shows an example float.

FIG. 6A shows a flowchart of an example method for separating a targetanalyte from a suspension.

FIG. 6B shows a flowchart of an example method for separating a targetanalyte from a suspension.

FIG. 7A shows an example clamp forming a seal between a float and atube.

FIG. 7B shows an example clamp forming a seal between a float and atube.

FIG. 8 shows a lowest density fraction being removed from an examplevessel after a suspension has undergone density-based separation.

FIG. 9 shows an example system for separating a target analyte.

FIG. 10A shows an example system for separating a target analyte.

FIG. 10B shows an example system for separating a target analyte.

DETAILED DESCRIPTION

This disclosure is directed to systems and methods for analyzing atarget analyte of a suspension. In one aspect, a system can be composedof a tube, a float, and a cap, the cap comprising a magnetic insert anda receiving piece. The system may also include a primary fluid to changethe location of the target analyte within the tube. The magnetic insertincludes a stopper and a magnet extending from the stopper; and, thereceiving piece, which is configured to hold the magnetic insert,includes a receiving stopper and a sheath. The sheath may includeimaging slides on opposite sides of the sheath. The cap introduces amagnetic field or a magnetic gradient to the tube to draw the targetanalyte bound to a particle to the cap. In another aspect, the cap mayinclude a stopper and an embedded magnet. The cap may include ananalysis piece on a bottom end of the stopper. In another aspect, thecap may include a fluid compartment and a filter at a bottom end of thestopper. The system, in another aspect, may include a separating fluidto separate non-target analytes from the target analyte.

Magnetic Cap

FIG. 1A shows an exploded view of an example magnetic cap 100. Themagnetic cap 100 includes a magnetic insert 102 and a receiving piece116, the receiving piece 116 configured to hold the magnetic insert 102,such that the magnetic insert 102 and the receiving piece 116 arecoaxial along a central axis 138. The magnetic insert 102 includes astopper 104 with a bottom end 112 and a top end 114. The magnetic insert102 also includes a magnet 106, the magnet 106 extending outward fromthe bottom end 112 of the stopper 104. The magnet 106 may be a permanentmagnet, such as a bar magnet or any other appropriately-shaped magnet.The magnetic insert 102 may also include an inlet port 108 and an outletport 110. The inlet port 108 and the outlet port 110 may, individually,extend the full length of the stopper 104 or may extend only a partiallength of the stopper 104. The inlet port 108 permits fluids to beintroduced into the system through the magnetic cap 100 without havingto remove the magnetic cap 100 from a vessel or without having topuncture the magnetic cap 100. The outlet port 110 permits fluids to beremoved from the system through the magnetic cap 100 without having toremove the magnetic cap 100 from the vessel or without having topuncture the magnetic cap 100. Alternatively, the magnetic cap 100 mayinclude only a single port that permits fluids to be both introduced toand removed from the vessel.

The receiving piece 116 includes a receiving stopper 118 with a bottomend 134 and a top end 136. The bottom end 134 is sized and shaped to fitwithin the vessel opening; the top end 136 may be sized and shaped toprevent the receiving piece 116 from sliding further into the vessel,such as by being greater in diameter (i.e. wider) than a diameter of aninner wall of the vessel opening. The receiving piece 116 also includesa sheath 124 that extends outward from the bottom end 134 of thereceiving stopper 118. The sheath 124 is hollow and is configured toreceive the magnet 106 of the magnetic insert 102. The sheath 124 may berectangular, hemispherical, triangular, conical, polyhedral, or anyappropriate shape. The receiving stopper 118 includes an opening 122configured to receive the bottom end 112 of the stopper 104 and a cavity120 configured to receive the top end 114 of the stopper 104. Thereceiving piece 116 may include an inlet opening 130 and an outletopening 132 at the bottom end 134. The inlet opening 130 and the outletopening 132 correspond to the inlet port 108 and the outlet port 110,respectively, of the magnetic insert 102, to permit fluids to flow intoand out of the vessel without being trapped within the receiving stopper118.

The receiving piece 116 may include imaging slides 126 and 128 locatedon opposite sides of the sheath 124. The imagining slides 126 and 128are smooth, thin, flat pieces of material that may be removably attachedto the sheath 124 by an adhesive, an oil, a gel, a grease, such asvacuum grease, a vacuum, or the like. The imaging slides 126 and 128 maybe reflective, opaque, transparent or translucent. The imaging slides126 and 128 may be composed of glass, plastic, metal, or combinationsthereof. For example, the imaging slides 126 and 128 can be a thinmicroscope slide disposed so that the imaging slides 126 and 128 may bedetached from the sheath 124 and placed onto a fluorescent microscope toimage for a target analyte. The imaging slides 126 and 128 may beaffixed to a frame or holder to facilitate ease of handling.

The magnetic cap 100 may be used to analyze a target analyte of asuspension. The target analyte, having been conjugated with a particleto form a target analyte-particle complex, may be attracted to and heldto one of the imaging slides 126 and 128 by a magnetic field or amagnetic gradient created by the magnet 106. The magnetic cap 100 maythen be removed from the vessel in which the magnetic cap 100 wasplaced. The imaging slides 126 and 128, with target analyte-particlecomplex held to the surface of the imaging slides 126 and 128, may beremoved from the sheath 126, and placed onto or within an imagingdevice, such as a fluorescent microscope, to detect or analyze thetarget analyte.

The particle may come in any form, including, but not limited to, abead, a nanoparticle (such as a quantum dot), a shaving, a filing, orthe like, such that the particle is capable of being attracted by amagnetic field or magnetic gradient introduced by a magnet. The particlemay itself be magnetic, diamagnetic, ferromagnetic, or paramagnetic.

FIG. 1B shows an exploded view of an example magnetic cap 140. Themagnetic cap 140 is similar to the magnetic cap 100, except that themagnetic cap 140 includes an electromagnet 142 instead of the magnet106. The electromagnet 142 includes a power source 144, such as abattery, DC or AC current supply, external to, disposed on or within thetop end 114 of the stopper 104, a first lead 146, a coil 148, a secondlead 150, and a core 152 around which the coil 148 is wrapped. Themagnetic cap 140 may also include a switch or control mechanism.

FIG. 1C shows an exploded view of an example magnetic cap 160. Themagnetic cap 160 is similar to the magnetic cap 100, except that themagnetic cap 160 includes a sheath 164 that is sized and shaped to fitflush against the sidewall of the vessel, such as a tube, therebypreventing any fluids from flowing between the sheath 164 and thesidewall of the vessel. The sheath 164 may be rectangular,hemispherical, triangular, conical, polyhedral, or any appropriateshape. The sheath 164 includes a flat facet 166 to hold the imagingslide 126 within a sample contained by the vessel. A magnet 162 may beshaped the same as the sheath 162 and sized proportionally to fit withinthe sheath 164. Alternatively, the magnet 162 may be any magnet capableof fitting within the sheath 164.

FIG. 2 shows an exploded view of an example magnetic cap 200. Themagnetic cap 200 includes a stopper 202 with a bottom end 206 and a topend 204. The magnetic cap 200 also includes a shaft 208, the shaft 208extending outward from the bottom end 206 of the stopper 202. Themagnetic cap 200 further includes a magnet 210 embedded within the shaft208. The magnet 210 may be a permanent magnet, such as a bar magnet orany other appropriately-shaped magnet. The magnetic cap 200 may alsoinclude an inlet port 214 and an outlet port 216 similar to the inletand outlet ports 108 and 110 as shown in FIG. 1A. It should be notedthat inlet port 214 is shown in FIG. 2 as extending only a partiallength of the stopper 202 and exiting at a sidewall of the stopper 202.The magnetic cap 200 also includes imaging slides 212 and 214 located onopposite sides of the shaft 208. The imaging slides 212 and 214 aresimilar to the imaging slides 126 and 128 as shown in FIG. 1A.

Alternatively, the shaft 208 may be sized and shaped to fit flushagainst the sidewall of the vessel, such as a tube, thereby preventingany fluids from flowing between the shaft 208 and the sidewall of thevessel. The shaft 208 may be rectangular, hemispherical, triangular,conical, polyhedral, or any appropriate shape. The shaft may include aflat facet to hold an imaging slide within a sample contained by thevessel. The magnet 210 may be shaped the same as the shaft 208 and sizedproportionally to be embedded within the shaft 208; or, the magnet 210may be any magnet capable of embedding within the shaft 208.

FIG. 3A shows an exploded view of an example magnetic cap 300. Themagnetic cap 300 includes a stopper 302 with a bottom end 308 and a topend 310. The bottom end 308 is sized and shaped to fit within an openingof a vessel. The top end 310 may be sized and shaped to prevent themagnetic cap 300 from sliding further into the vessel, such as by beinggreater in diameter (i.e. wider) than a diameter of an inner wall of thevessel. The magnetic cap 300 also includes a cap magnet 304. The capmagnet 304 may be embedded within the stopper 302 or may be attached tothe bottom end 308 of the stopper 302 with an adhesive, an oil, a gel, agrease, or the like. The magnetic cap 300 may also include an analysispiece 306, the analysis piece 306 being removably attached to the bottomend 308 of the stopper 302 or to the cap magnet 304 via an adhesive, anoil, a gel, a grease, such as vacuum grease, a vacuum, or the like. Theanalysis piece 306 is a smooth, thin, flat piece of material that may beremoved from the stopper 302 or the cap magnet 304. The analysis piece306 may be reflective, opaque, transparent or translucent. The analysispiece 306 may be composed of glass, plastic, metal, or combinationsthereof. For example, the analysis piece 306 can be a thin microscopeslide disposed so that the analysis piece 306 may be detached from thestopper 302 and placed onto a fluorescent microscope to image for atarget analyte. The bottom end 308 of the magnetic cap 300 may beperpendicular with respect to a central axis 312 of the magnetic cap300, as shown in FIG. 3A; or, the bottom end 308 may be angled withrespect to the central axis 312 of the magnetic cap 320, as shown inFIG. 3B, such that a first edge 322 of the bottom end 308 is longer thana second edge 324 of the bottom end 308. The angled bottom end 308 maydecrease or inhibit the formation or presence of air bubbles on theanalysis piece 306.

FIG. 3C shows an exploded view of an example magnetic cap 330. Themagnetic cap 330 is similar to magnetic cap 300 except that magnetic cap330 includes an inlet port 332 and an outlet port 334. The inlet port332 and the outlet port 334 are similar to the inlet and outlet ports214 and 216 as shown in FIG. 2.

FIG. 3D shows an exploded view of an example magnetic cap 340. Themagnetic cap 340 is similar to the magnetic cap 300, except that themagnetic cap 340 includes an electromagnet 342 instead of the magnet306. The electromagnet 342 includes a power supply 344, such as abattery, DC or AC current supply, external to disposed on or within thetop end 310 of the stopper 302, a first lead 346, a coil 348, a secondlead 350, and a core 352 around which the coil 348 is wrapped. Themagnetic cap 340 may also include a switch or control mechanism.

FIG. 3E shows an exploded view of an example magnetic cap 360. Themagnetic cap 360 is similar to the magnetic cap 3B, except that themagnetic cap 360 includes an inlet/outlet port 364 to add or removefluids and an ejection port 366 to accept an ejection prong (not shownto eject the analysis piece 306 from the bottom end 308 of the stopper302. The ejection port 366 may extend from the top end 310 to the bottomend 308 of the stopper 302; or, the ejection port 366 may partiallyextend through the stopper 302, thereby extending from a sidewall of thestopper 302 to the bottom end 308 of the stopper 302. The magnetic cap360 also includes a magnet 362.

FIG. 4A shows an exploded view of an example magnetic cap 400. The cap400 includes a stopper 402 with a bottom end 414 and a top end 416. Thebottom end 414 is sized and shaped to fit within an opening of a vessel.The top end 416 may be sized and shaped to prevent the magnetic cap 400from sliding further into the vessel, such as by being greater indiameter (i.e. wider) than a diameter of an inner wall of the vessel.The cap 400 also includes a cap magnet 404, a fluid compartment 410, anda filter 406. The cap magnet 404 may be embedded within the stopper 402.The filter 406 includes pores 408 that are sized to allow unboundparticles (i.e. particles not conjugated to any component of thesuspension) to pass through the filter 406 and into the fluidcompartment 410. The fluid compartment 410 is a cavity—between thebottom end 414 of the stopper 402 and the filter 406—formed by extendinga sidewall 412 from the bottom end 414 of the stopper 402.

The magnetic cap 400 may be used to analyze a target analyte of asuspension. The filter 406 may also be configured to trap or hold thetarget analyte. The target analyte may be trapped within one of thepores 408 or may be held to the surface of the filter 406. The magneticcap 400 may then be removed from a vessel.

A filter end 418 of the magnetic cap 400 may be perpendicular withrespect to a central axis 420 of the magnetic cap 400, as shown in FIG.4A; or, the filter end 418 may be angled with respect to the centralaxis 420 of the magnetic cap 422, as shown in FIG. 4B, such that a firstedge 432 of the filter end 418 is longer than a second edge 434 of thefilter end 418. The angled filter end 418 may decrease or inhibit theformation or presence of air bubbles on the filter 428.

FIG. 4C shows an isometric view of a magnetic cap 440. The magnetic cap440 is similar to the magnetic cap 400, except the magnetic cap 440includes more than one filter 446 and 452. The filters 446 and 452 maybe stacked successively (i.e. touching) or may be separated—therebyforming a repository 450 between the filters 446 and 452 and a fluidcompartment 444 between the bottom end 414 of the stopper 402 and theuppermost filter 446. The fluid compartment 444 and the repository 450may collect different analytes based on the size of the pores 448 and454 in the filters 446 and 452, respectively.

The magnet may be, but is not limited to, a ring magnet, a bar magnet, ahorseshoe magnet, a spherical magnet, a polygon-shaped magnet, apolyhedral-shaped magnet, a wand magnet, a kidney-shaped magnet, atrapezoidal magnet, a disk magnet, a cow magnet, a block or brickmagnet, an electromagnet, and a switchable magnet.

The magnet may be permanently embedded or removably embedded within thestopper.

Magnetic Cap and Vessel System

The magnetic cap may be used in a system for separating a suspensionsuspected of containing a target analyte, the system including a vessel,the magnetic cap, and a primary fluid. The vessel is configured to holda fluid, a suspension, a solution, or the like. Suppose, for example,the suspension includes three fractions. During centrifugation, thesuspension may be divided into and settle into the three fractions,including a high density fraction, a medium density fraction, and a lowdensity fraction. The primary fluid is a liquid substance that has agreater density than the density of the medium density fraction, thoughthe primary fluid may have a density greater than the high densityfraction. The primary fluid moves below the medium density fraction,thereby moving the medium density fraction upwards within the vessel.The system may also include a separating fluid, the separating fluidbeing a liquid substance that has a density that is less than thedensity the medium density fraction. The separating fluid inhibitsnon-target analytes from passing through towards the magnetic cap. Theweak magnetic attraction may not overcome the force required to drag thenon-target analytes through the separating fluid. However, the targetanalyte, which may be bound to particles attracted to the magnetic capby stronger, more specific interactions, by, for example, a strongnon-covalent interaction between complementary molecules, such as biotinand streptavidin, is capable of passing through the separating fluid.For example, the surface tension may break the weak bonds between thenon-target analyte and the particle; or, the viscosity of the separatingfluid may be great to inhibit passage of the weakly-bound non-targetanalyte. A plurality of primary fluids may also be used. The pluralityof primary fluids may be used, for example, such that multiple densitylayers are formed or whereby each primary fluid is used sequentially toseparate a suspension fraction, draw the target analytes out of thefraction, remove the separated suspension fraction, and repeat towithdraw individual target analytes from individual suspension layers.

The compositions of the primary fluid and the separating fluid may beselected so that suspension fractions and suspension fluid areimmiscible in and inert with respect to the primary fluid and theseparating fluid. Because the primary fluid and the separating fluid areimmiscible in the suspension fractions and suspension fluid, the primaryfluid and the separating fluid do not mix with the suspension fractionsor the suspension fluid, which prevents a change in the density of thefluids and prevents a change in the density gradient within the layeredsuspension materials. Examples of suitable primary fluids include, butare not limited to, solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol, cesium chloride, sucrose, sugar-based solutions,polymer-based solutions, surfactants, dextran, fluorinated liquids, suchas perfluoroketones, perfluorocyclopentanone, perfluorocyclohexanone,fluorinated ketones, hydrofluoroethers, hydrofluorocarbons,perfluorocarbons, and perfluoropolyethers; silicon and silicon-basedliquids, such as phenylmethyl siloxane.

Examples of suitable separating fluids include, but are not limited to,an organic solvent, a liquid wax, an oil, a gas, and combinationsthereof; olive oil, mineral oil, silicone oil, chill-out liquid wax,paraffin wax, microcrystalline waxes, soy and palm waxes, candle waxes,thermoset waxes, hot melt adhesives, atactic polypropylene andpolyolefin compounds, petroleum waxes, dental waxes, animal waxes,vegetable waxes, mineral waxes, petroleum waxes, and synthetic waxes,such as ethylenic polymers, chlorinated naphthalenes or hydrocarbon-typewaxes; immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, and combinations thereof; organic solvents suchas 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol,cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methylether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane,propylene carbonate, tetramethylene sulfones, and ionic liquids.

Examples of suitable vessels include, but are not limited to, a tube, awell, a bottle, a flask, a beaker, a column, and a microfluidic device.

The system may also include a solution containing the particle toconjugate with the target analyte to form a target analyte-particlecomplex, such that when the magnetic cap is added to the vessel, thetarget analyte-particle complex is attracted to the magnetic cap.

Float and Tube System

The magnetic cap may be used in combination with a float and tubesystem. The primary fluid, the separating fluid, and the solutioncontaining particles configured to bind to the target analyte may alsobe used in combination with the float and tube system. FIG. 5A shows anisometric view of an example tube and float system 500. The system 500includes a tube 502 and a float 504 suspended within a suspension 506.In the example of FIG. 5A, the tube 502 has a circular cross-section, afirst closed end 508, and a second open end 510. The open end 510 issized to receive a sealing cap 512. The tube may also have two open endsthat are sized to receive sealing caps, such as the example tube andfloat system 520 shown FIG. 5B. The system 520 is similar to the system500 except the tube 502 is replaced by a tube 522 that includes two openends 524 and 526 configured to receive the sealing cap 512 and a sealingcap 528, respectively. The tubes 502 and 522 have a generallycylindrical geometry, but may also have a tapered geometry that widens,narrows, or a combination thereof toward the open ends 510 and 524,respectively. Although the tubes 502 and 522 have a circularcross-section, in other embodiments, the tubes 502 and 522 can haveelliptical, square, triangular, rectangular, octagonal, or any othersuitable cross-sectional shape that substantially extends the length ofthe tube. The tubes 502 and 522 can be composed of a transparent orsemitransparent flexible material, such as flexible plastic or anothersuitable material. The tube 502 may also include a plug 514, as seen inmagnified view 516, at the closed end 508 to permit the removal of afluid, the suspension, or a suspension fraction, whether with a syringe,a pump, by draining, or the like. The tube 502 may have a sidewall and afirst diameter. The float 504 can be captured within the tube by aninterference fit.

FIG. 5C shows an isometric view of the float 504 shown in FIGS. 5A and5B. The float 504 includes a main body 530, two teardrop-shaped end caps532 and 534, and support members 536 radially spaced and axiallyoriented on the main body 530. The float can also include twodome-shaped end caps or two cone-shaped end caps or anyappropriately-shaped end cap. The support members 536 provide a supportengagement with the inner wall of the tube 502. The support members 536may extend the full length of the main body 530 or may extend a partiallength of the main body 530, thereby providing an area on the main body530 with no support member located at any circumferential point on thefloat 504. Alternatively, the support members 536 may be capable ofbeing compressed by introducing an external force, such as by a clamp.

In alternative embodiments, the number of support members, supportmember spacing, and support member thickness can each be independentlyvaried. The support members 536 can also be broken or segmented. Themain body 530 is sized to have an outer diameter that is less than theinner diameter of the tube 502, thereby defining fluid retentionchannels between the outer surface of the main body 530 and the innerwall of the tube 502. The surfaces of the main body 530 between thesupport members 536 can be flat, curved or have another suitablegeometry. In the example of FIG. 5C, the support members 536 and themain body 530 form a single structure. The support members 536 and themain body 530, alternatively, may be separate structures.

Embodiments include other types of geometric shapes for float end caps.The top end cap may be teardrop-shaped, dome-shaped, cone-shaped, or anyother appropriate shape. The bottom end cap may be teardrop-shaped,dome-shaped, cone-shaped, or any other appropriate shape. In otherembodiments, the main body of the float 504 can include a variety ofdifferent support structures for separating target materials, supportingthe tube wall, or directing the suspension fluid around the float duringcentrifugation. Embodiments are not intended to be limited to theseexamples. The main body may include a number of protrusions that providesupport for the tube. In alternative embodiments, the number and patternof protrusions can be varied. The main body may include a singlecontinuous helical structure or ridge that spirals around the main bodycreating a helical channel. In other embodiments, the helical ridge canbe rounded or broken or segmented to allow fluid to flow betweenadjacent turns of the helical ridge. In various embodiments, the helicalridge spacing and rib thickness can be independently varied. In anotherembodiment, the main body may include a support member extendingradially from and circumferentially around the main body. In anotherembodiment, the support members may be tapered.

The float can be composed of a variety of different materials including,but not limited to, metals; organic or inorganic materials; ferrousplastics; sintered metal; machined metal; plastic materials andcombinations thereof.

The sealing cap may be composed of a variety of different materialsincluding, but not limited to, organic or inorganic materials; plasticmaterials; and combination thereof.

The end caps of the float may be manufactured as a portion of the mainbody, thereby being one singular structure, by machining, injectionmolding, additive techniques, or the like; or, the end caps may beconnected to the main body by a press fit, an adhesive, a screw, anyother appropriate method by which to hold at least two pieces together,or combinations thereof.

The plug 514 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access contents of the tube 502 interior andre-seals when the needle or implement is removed. The plug 514 can beformed in the openings and/or the bottom interior of the tube usingheated liquid rubber that can be shaped and hardens as the rubber cools.The adhesive used to attach the plug 514 to the wall of the opening andtube interior and can be a polymer-based adhesive, an epoxy, a contactadhesive or any other suitable material for bonding rubber to plastic orcreating a thermal bond.

Methods

FIGS. 6A and 6B show flow diagrams of example methods for separating atarget analyte from a suspension. Referring now to FIG. 6A, in block602, a suspension is obtained and then added to a vessel, such as atube. In block 604, a float is added to the vessel and a sealing capseals the vessel. FIG. 5A shows the suspension 506, such as blood, addedto a vessel, such as the tube 502. The float 504 is then added to thetube 502 and the sealing cap 512 is added to the tube 502 to seal theopening 508. A sample suspension can be urine, blood, bone marrow,cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, nippleaspirate fluid, saliva, amniotic fluid, vaginal secretions, mucusmembrane secretions, aqueous humor, vitreous humor, vomit, and any otherphysiological fluid or semi-solid. It should also be understood that atarget analyte can be a cell, such as ova or a circulating tumor cell(“CTC”), a circulating endothelial cell, a fetal cell, a nucleated redblood cell, a red blood cell, a vesicle, a liposome, a protein, anucleic acid, a biological molecule, a naturally occurring orartificially prepared microscopic unit having an enclosed membrane,parasites, microorganisms, viruses, or inflammatory cells.

Returning to FIG. 6A, in block 606, the float, the tube, and thesuspension undergo density-based separation, such as by centrifugation,thereby permitting separation of the suspension into density-basedfractions along an axial position in the tube based on density. In block608, a clamp is applied to the system.

FIG. 7A shows an isometric view of the tube and float system 500 havingundergone density-based separation, such as by centrifugation. Suppose,for example, the suspension includes three fractions. The suspensionseparates into three fractions, with a highest density fraction 703located on the bottom, a lowest density fraction 701 located on top, anda medium density fraction 702 located in between. The float 504 may haveany appropriate density to settle within one of the fractions. Thedensity of the float 504 can be selected so that the float 504 settlesat the same axial position of the target analyte. The target analyte canbe trapped within an analysis area between the float 504 and the tube502.

After the suspension is separated into fractions 701-703, a seal may beformed between the tube 502 and the float 504. For example, as shown inFIG. 7A, a clamp 704 may be placed around the tube 502 at the interfacebetween the medium density fraction 702 and the highest density fraction703 to form the seal. The clamp 704 may be, but is not limited to, acompressible metal ring, a collet clamp, an O-ring, a pipe clamp, a hoseclamp, a spring clamp, a strap clamp, a tie, such as a zip tie, or apiezoelectric ring. The clamp 704 may or may not include a thermalelement, such as a heated wire, to soften the tube 502. The clamp 704circumferentially applies pressure directed toward the central axis ofthe tube 502. The inward circumferential pressure causes the tube 502 tocollapse inwardly. The clamp 704 forces the inner wall of the tube 502against the main body 530 of the float 504 essentially forming a sealbetween the medium density fraction 702 and the highest density fraction703, as shown in magnified cross-sectional view 706. The seal inhibitsfluid flow between the medium density fraction 702 and the highestdensity fraction 703 and holds the float 504 in place against thebuoyant forces exerted by the highest density fraction 703.Alternatively, the seal may be formed by ultrasonically welding the tube502 to the float 504. Alternatively, the seal may be formed by meltingthe tube 502 to the float 504.

FIG. 7B shows a collet clamp 712 placed on the tube 502. Magnified view718 shows a cross-sectional view across a system with the collet clamp712. The collet clamp 712 is a clamp having an inner collar 716 and anouter collar 714, the outer collar 714 being capable of receiving theinner collar 716. The inner collar 716 may be segmented. The outer andinner collars 714 and 716 include complementary threads. The outer andinner collars 714 and 716 both include a cavity to receive the tube 502.The threads of the outer collar 714 engage the threads of the innercollar 716, and screwing the outer collar 714 further onto the innercollar 716 causes the inner collar 716 to compress. When the colletclamp 712 is placed around the tube 502 and the outer collar 714 isscrewed onto the inner collar 716, the inner collar 716 compresses,thereby applying uniform pressure circumferentially on the tube to causethe tube to compress towards the float 504 and creating a seal.

Returning to FIG. 6A, in block 610, the lowest density fraction isremoved from the tube. FIG. 8 shows the lowest density fraction 701being removed using an extraction device 802. The extraction device 802may be a pump or syringe. With the clamp 704 in place, the lowestdensity fraction 701 can be removed by pouring off, pipetting,aspirating, or pumping. Alternatively, the lowest density fraction 701may be removed prior to the clamp 704 clamping the tube 502.

Returning to FIG. 6A, in block 612, the primary fluid is added to thetube and the system is then re-centrifuged to wash the target analyteoff of the tube and float surfaces and re-suspend the target analyte. Inblock 614, the solution including the particle to conjugate to thetarget analyte to form the target analyte-particle complex is added tothe tube and incubated. In block 616, the magnetic cap is inserted andthe target analyte-particle complex is drawn to the magnetic cap. Inblock 618, the magnetic cap is removed.

FIG. 9 shows the magnetic cap 100 inserted into the tube 502. Themagnetic cap 100 may be inserted after a primary fluid 902 has beenadded to the tube 502, the system has been re-centrifuged, and asolution including a particle 910 to conjugate to the target analyte 908to form a target analyte-particle complex 906 has been added. Theprimary fluid 902, having a density greater than the medium densityfraction 702, causes the medium density fraction 702 to move upwardswithin the tube 502 after re-centrifugation. The seal formed by theclamp 704 prevents any fluids, including the highest density fraction703, from moving past the clamp 704. The float 504 does not move furtherdown in the tube 502 due to the clamp 704. The target analyte-particlecomplex 906 includes the target analyte 908 and the particle 910. Theparticle 910 is configured to be attracted to the magnetic field ormagnetic gradient introduced by a magnet, such as the magnet 106described above with reference to FIG. 1. The target analyte-particlecomplex 906 is then drawn to the magnet 106 of the magnetic cap 100. Thetarget analyte-particle complex 906 is held to the sheath 124 and themagnetic cap 100 is then removed from the tube 502. The magnetic cap 100may be inserted into a vessel including a solution, such as a buffer,and the magnetic insert 102 may then be removed from the receiving piece116, thereby removing the target analyte-particle complex 906 from thesheath 124. The target analyte-particle complex 906 may then undergofurther processing or testing.

Alternatively, the solution including the particle to conjugate to thetarget analyte to form the target analyte-particle complex may be addedbefore re-centrifugation.

Before the target analyte-particle is attracted to the magnetic cap 100,magnets may be placed externally to the tube to draw the targetanalyte-particle complex to the sidewall of the tube. The externalmagnets may be placed on opposite sides of the tube 502 to draw thetarget analyte-complex 906 to the sidewall of the tube 502.Alternatively, a single magnet may be used to draw the targetanalyte-particle complex 906 to one side of the tube 502. Alternatively,a single donut- or ring-shaped magnet may encircle the tube 502 to drawthe target analyte-particle complex 906 to the closest side of the tube502. Alternatively, more than two magnets may be used to draw the targetanalyte-complex 906 to the closest side of the tube 502. The externalmagnets may be, but are not limited to, ring magnets, bar magnets,horseshoe magnets, spherical magnets, polygon-shaped magnets,polyhedral-shaped magnets, wand magnets, kidney-shaped magnets,trapezoidal magnets, disk magnets, cow magnets, block or brick magnets,electromagnets, and switchable magnets.

Referring now to FIG. 6B, blocks 602-610 refer to the same operationsdescribed above with reference to FIG. 6A. In block 620, the solutionincluding the particle to conjugate to the target analyte to form thetarget analyte-particle complex is added to the tube and incubated. Inblock 622, the primary fluid and a separating fluid are added to thetube, and the system is then re-centrifuged. In block 624, the magneticcap is inserted and the target analyte-particle complex is drawn to themagnetic cap. In block 626, the magnetic cap is removed from the tube.

FIG. 10A shows the target analyte-particle complex 906 drawn to themagnetic cap 300 after the primary fluid 902 and a separating fluid 1002are added to the tube 502 and after the system is re-centrifuged. Thesealing cap 512 is removed from the tube 502 and the magnetic cap 300 isthen inserted into the tube 502. The magnet 304 of the magnetic cap 300creates a magnetic field or a magnetic gradient strong enough to drawthe target analyte-complex 906 through the separating fluid 1002 to theanalysis piece 306. Alternatively, an external magnet may be broughtproximal to an outer wall of the tube 502 at a height substantially thesame as the location of the medium density fraction 702 within the tube502. The external magnet can be moved upwards towards the magnetic cap300 and past the separating fluid 1012, which causes the targetanalyte-particle complex 906 move upward within the tube 502 towards themagnetic cap 300 as the external magnet moves upwards along the outsideof the tube 502.

The primary fluid 902, having a density greater than the medium densityfraction 702, displaces the medium density fraction 702, thereby causingthe medium density fraction 702 to move upwards within the tube 502after re-centrifugation. The separating fluid 1002, having a densityless than the medium density fraction 702, sits on top of the mediumdensity fraction 702. As shown in magnified view 1006, the separatingfluid 1002 inhibits the non-target analytes 1004 from passing throughthe separating fluid 1002 and being held to the analysis piece 306, asthe separating fluid 902 may break the weak bonds between the non-targetanalyte 1004 and the particle 910 so that the non-target analyte 1004does not travel towards the cap magnet 304 and to the analysis piece306. The weak magnetic attraction may not overcome the force required todrag the non-target analytes 1004 through the separating fluid 1002.However, the target analyte 908, which may be bound to the magneticparticles by stronger, more specific interactions, by, for example, astrong non-covalent interaction between complementary molecules, such asbiotin and streptavidin, is capable of passing through the separatingfluid 1002. The float 504 does not move further down in the tube 502also due to the clamp 704. The seal formed by the clamp 704 prevents anyfluids, including the highest density fraction 703, from moving passedthe clamp 704 in any direction.

The magnetic cap 300 may then be removed from the tube 502. The analysispiece 306 may then be separated from the magnetic cap 300 and placed onor within an imaging device, such as a microscope, to analyze the targetanalyte; or, the analysis piece 306 may be further processed forsubsequent analysis of the target analyte.

FIG. 10B shows the target analyte-particle complex 906 drawn to themagnetic cap 400 after the primary fluid 902 and a separating fluid 1002are added to the tube 502 and after the system is re-centrifuged. Thesealing cap 512 is removed from the tube 502 and the magnetic cap 400described above with reference to FIG. 4 is then inserted into the tube502. The magnet 404 of the magnetic cap 400 creates a magnetic field ora magnetic gradient that draws the target analyte-particle complex 906through the separating fluid 1002 to the filter 406. The particle 910may be unbound and therefore may pass through the pores 408 of thefilter 406. The unbound particle 910 then collects in the fluidcompartment 410 of the magnetic cap 400. The target analyte-particlecomplex 906 may then be trapped within or around the pore 408.Alternatively, an external magnet may be brought proximal to an outerwall of the tube 502 substantially the same as the location of themedium density fraction 702 within the tube 502. As the external magnetis moved upwards towards the magnetic cap 400 and passed the separatingfluid 1002, moves upwards within the tube 502 towards the magnetic cap400.

The primary fluid 902, having a density greater than the medium densityfraction 702, displaces the medium density fraction 702, thereby causingthe medium density fraction 702 to move upwards within the tube 502after re-centrifugation. The separating fluid 1002, having a densityless than the medium density fraction 702, sits on top of the mediumdensity fraction 1002. As seen in magnified view 1008, the separatingfluid 1002 inhibits the non-target analytes 1004 from passing throughthe separating fluid 1002 to the filter 406, as the separating fluid1002 may break the weak bonds between the non-target analyte 1004 andthe particle 910 so that the non-target analyte 1004 does not traveltowards the cap magnet 404. The weak magnetic attraction may notovercome the force required to drag the non-target analytes 1004 throughthe separating fluid 1002. However, the target analyte 908, which may bebound to the magnetic particles by stronger, more specific interactions,by, for example, a strong non-covalent interaction between complementarymolecules, such as biotin and streptavidin, is capable of passingthrough the separating fluid 1002. The float 504 does not move furtherdown in the tube 502 also due to the clamp 704. The seal formed by theclamp 704 prevents any fluids, including the highest density fraction703, from moving passed the clamp 704 in any direction.

The magnetic cap 400 may then be removed from the tube 502. The filter406 may then be separated from the magnetic cap 400 and processed toremove the target analyte-particle complex 906; or, the targetanalyte-particle complex 906 may be flushed out of the pore 408.

After the magnetic cap has been removed, the magnetic cap may be washedto remove unwanted material or particles from the cap. The wash mayoccur by spraying or rinsing the cap with a wash solution.Alternatively, the wash may be performed by immersing the cap into acontainer having a wash solution. A magnetic particle may be cleavedfrom a target analyte during the washing step by proteolytic cleavage,pH variation, or salt concentration variation (i.e. increasing the saltconcentration of the surrounding solution to disrupt the molecularinteractions that hold the target analyte to the magnetic particle). Thetarget analyte may also be processed directly on the magnetic cap.

Alternatively, a sealing ring may be used to maintain the seal betweenthe tube and the float so that clamp may be removed. The sealing ringmay be placed between the clamp and the tube, and then tightened,thereby causing the tube to constrict and form the seal with the float.The sealing ring remains tightened and in tension. Alternatively, noclamp may be required to apply a uniform circumferential force, such aswith a sealing ring composed of a piezoelectric material. Applying anelectric potential to the sealing ring produces a mechanical strain,thereby causing the sealing ring to tighten and constrict the tube toform the seal between the tube and the float.

A solution containing a fluorescent probe may be used to label thetarget analyte, thereby providing a fluorescent signal foridentification and characterization. The solution containing thefluorescent probe may be added to the suspension before the suspensionis added to the vessel, after the suspension is added to the vessel butbefore centrifugation, or after the suspension has undergonecentrifugation. The fluorescent probe includes a fluorescent moleculebound to a ligand. The target analyte may have a number of differenttypes of surface markers. Each type of surface marker is a molecule,such an antigen, capable of attaching a particular ligand, such as anantibody. As a result, ligands can be used to classify the targetanalyte and determine the specific type of target analytes present inthe suspension by conjugating ligands that attach to particular surfacemarkers with a particular fluorescent molecule. Examples of suitablefluorescent molecules include, but are not limited to, quantum dots;commercially available dyes, such as fluorescein, FITC (“fluoresceinisothiocyanate”), R-phycoerythrin (“PE”), Texas Red, allophycocyanin,Cy5, Cy7, cascade blue, DAPI (“4′,6-diamidino-2-phenylindole”) and TRITC(“tetramethylrhodamine isothiocyanate”); combinations of dyes, such asCY5PE, CY7APC, and CY7PE; and synthesized molecules, such asself-assembling nucleic acid structures. Many solutions may be used,such that each solution includes a different type of fluorescentmolecule bound to a different ligand.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents:

We claim:
 1. A cap to be received by an open end of a vessel, the capcomprising: a stopper including a bottom end and a top end; a magnetembedded within the stopper; and an analysis piece removably attached tothe bottom end of the stopper, wherein the analysis piece is a coverslip or a slide.
 2. The cap of claim 1, wherein the bottom end is sizedand shaped to fit within an opening of the vessel and wherein the topend is wider than the opening of the vessel.
 3. The cap of claim 1,wherein the bottom end is angled.
 4. The cap of claim 1, furthercomprising at least one port to permit fluids to be introduced to andremoved from the vessel.
 5. The cap of claim 1, wherein the magnet isremovably embedded within the stopper.
 6. The cap of claim 1, whereinthe magnet is permanently embedded within the stopper.
 7. The cap ofclaim 1, further comprising an extraction port to receive an extractionprong to eject the analysis piece from the bottom end, the extractionport extending at least partially through the stopper.
 8. A cap to bereceived by an open end of a vessel, the cap comprising: a stopperincluding a bottom end and a top end; a magnet embedded within thestopper; a sidewall extending downwardly from the bottom end to form afilter end; at least one filter in the filter end; and a fluidcompartment located between the at least one filter and the bottom end,the at least one filter including at least one pore.
 9. The cap of claim8, further comprising two or more filters, and wherein the fluidcompartment is located between the bottom end of the stopper and thefilter located closest to the bottom end of the stopper.
 10. The cap ofclaim 9, wherein the two or more filters are stacked.
 11. The cap ofclaim 9, wherein the two or more filters are separated to form arepository between consecutive filters.
 12. A cap to be received by anopen end of a vessel, the cap comprising: a stopper including a bottomend and a top end; a magnet embedded within the stopper; and an inletport to permit fluids to be introduced to the vessel and an outlet portto permit fluids to be removed from the vessel.
 13. A cap to be receivedby an open end of a vessel, the cap comprising: a magnetic insert, themagnetic insert comprising: a stopper including a bottom end and a topend; and, a magnet extending outwardly from the bottom end, wherein thebottom end is sized and shaped to fit within an opening of the vessel;and, a receiving piece, the receiving piece comprising: a receivingstopper including a bottom end and a top end; and, a sheath to receivethe magnet, the sheath extending outwardly from the bottom end, thebottom end to fit within the opening of the vessel, the receivingstopper to receive the bottom end of the stopper of the magnetic insert.14. The cap of claim 13, the receiving piece further comprising a firstslide removably attached to a first side of the sheath.
 15. The cap ofclaim 14, the receiving piece further comprising a second slideremovably attached to a second side of the sheath, wherein the first andsecond sides are opposite each other.
 16. The cap of claim 13, whereinthe top end of the stopper and the top end of the receiving stopper arewider than the opening of the vessel.
 17. The cap of claim 13, whereinthe magnet is a permanent magnet or an electromagnet.
 18. The cap ofclaim 13, wherein the magnetic insert further comprises at least oneport and the receiving piece comprises at least one openingcorresponding to the at least one port to permit fluids to be introducedto and removed from the vessel.
 19. The cap of claim 13, the magneticinsert further comprising an inlet port to permit fluids to beintroduced to the vessel and an outlet port to permit fluids to beremoved from the vessel; and the receiving piece further comprising aninlet opening corresponding to the inlet port and an outlet openingcorresponding to the outlet port.
 20. The cap of claim 13, wherein thesheath is sized and shaped to fit flush against a sidewall of thevessel.
 21. The cap of claim 13, wherein the sheath is hemispherical,triangular, or rectangular.